Journal of the Science of Food and Agriculture J Sci Food Agric 86:1926–1931 (2006)

Analysis of pesticide residues by onlinereversed-phase liquid chromatography–gas chromatography in the oil from olivesgrown in an experimental plot. Part IIRaquel Sanchez,1 Jose Manuel Cortes,2 Ana Vazquez,2 Jose Villen-Altamirano3 andJesus Villen1∗1Escuela Tecnica Superior de Ingenieros Agronomos, Universidad de Castilla–La Mancha, Campus Universitario s/n, 02071 Albacete,Spain2Escuela Universitaria de Magisterio de Albacete, Departamento de Quımica-Fısica, Universidad de Castilla–La Mancha, CampusUniversitario s/n, 02071 Albacete, Spain3Escuela Universitaria de Informatica, Universidad Politecnica de Madrid, Departamento de Matematica Aplicada, Calle arboleda s/n,28031 Madrid, Spain

Abstract: The effect of the pesticide dose used to control pests in olive trees and the date of treatment on theresidues present in the oil were studied for four organophosphorus pesticides (diazinon, malathion, trichlorphonand chlorfenvinphos) and one organochlorine (endosulfan). Pesticide residue analysis was performed usingonline reversed-phase liquid chromatography–gas chromatography, using an automated through oven transferadsorption desorption interface and selective detectors, such as nitrogen–phosphorus detector and electroniccapture detector. A simple filtration step was necessary before the chromatographic analysis of samples. Theobtained data were statistically analyzed and conclusions about olive pesticide treatments are presented. 2006 Society of Chemical Industry

Keywords: online coupling RPLC-GC; automated TOTAD interface; pesticide residue analysis; olive oil;experimental treatments

INTRODUCTIONOlive oil has been used for hundreds of years asnutritional food, as medication and as a cosmetic.Extra-virgin oil, particularly, possesses characteristicproperties that distinguish it from other vegetableoils. Some of those properties make this productan important element in the ‘Mediterranean diet’.Consumption is increasing thanks to its excellentorganoleptic and nutritive qualities and to growingconsumer preference for minimally processed foods.1

Pesticides are applied to olive trees in order toprotect the crop from attack by pests and the presenceof weeds. Most of the products used to control themare organophosphorus insecticides, the toxicologicaleffects of which are well known. The residues ofthese compounds may remain and contaminate the oilproduced and so it is desirable to keep toxic residues aslow as possible because of the high dietary and healthvalue of olive oil. Maximum residue limits (MRLs) forseveral pesticides have been fixed by the FAO/WHOCodex Committee for olives and olive oil2 and thesevary from 10 to 0.5 mg L−1.

The most commonly used methods for the analysisof pesticide residues in olive oil are multi-residuemethods for fatty substrates, based on partitioning

between hexane or light petroleum and acetonitrile,and gas chromatography (GC) analysis.3 However,these methods need large amounts of toxic andcontaminant solvents, and much time is spent onthe sample preparation steps, which are very tedious.An alternative to such traditional techniques is to useliquid chromatography (LC),4 while LC coupled toonline gas chromatography (LC-GC) has become apowerful tool for the trace-level analysis of complexmatrices such as olive oil.5

Research into pesticide residues in olive oil hasestablished that residue concentrations in oil arerelated to the number of treatments, the pre-harvestinterval and the fat solubility of the compoundsused.3

In a previous work, our research group stud-ied the effect of dose and date of treatmenton the residue levels found in oil for some ofthe most frequently used organophosphorus pesti-cides (dimethoathe, methidathion, chlorpyriphos andfenitrothion). The olive oil samples were analysedby a uni-residue method by online reversed-phaseliquid chromatography–gas chromatography with anflame ionization detector (RPLC-GC-FID), usingan automated TOTAD interface.6 The analytical

∗ Correspondence to: Jesus Villen, Escuela Tecnica Superior de Ingenieros Agronomos, Universidad de Castilla–La Mancha, Campus Universitario s/n, 02071Albacete, SpainE-mail: jesus.villen@uclm.esContract/grant sponsor: Comision Interministerial de Ciencia y Tecnologıa (CICYT), Spain; contract/grant number: PTR 1995-0626-OP(Received 9 March 2005; revised version received 27 July 2005; accepted 7 December 2005)Published online 17 July 2006; DOI: 10.1002/jsfa.2565

2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00

Analysis of pesticide residues in olive oil. Part II

method was previously developed by our researchgroup.7 Some limitations in this previous studywere the few olive trees treated and the useof FID in the analysis of the olive oil sam-ples, although valuable conclusions were neverthelessobtained.

The aim of the present work was both to amplifythe study carried out previously by including afurther four organophosphorus pesticides (diazinon,malathion, trichlorphon and chlorfenvinphos) andone organochlorine pesticide (endosulfan) and totest the RPLC-GC analytical methods using selectivedetectors. In the present study the number ofolive trees treated was higher than in the previousstudy in order to minimize the variability of theresults. The trees were sprayed with a mixture ofthe pesticides and the analysis of organophosphoruspesticides was carried out by online RPLC-GC-NPD using the automated TOTAD interface andthe multi-residue method developed previously.8

Endosulfan was analysed by online RPLC-GC-ECDusing the method developed to this end in the presentwork.

MATERIALS AND METHODSField trialThe field trial was carried out in the same olivegrove as the previous study,6 although in this caseolive trees were grouped into plots of four trees tominimize variability. A mixture of the pesticides usedwas applied to 24 plots. One plot, considered as ablank, was not treated. Treatments at different dosesand on different dates were carried out in the olivegrove in July and November of 2003. Eight plots weretreated at the recommended dose in July, eight plotsat the recommended dose in July and November,and eight plots at twice the recommended dose inJuly and November. The recommended doses areobtained by diluting, in 100 L: 120 mL of diazinon60% weight/volume (w/v), 300 mL of malathion 50%w/v, 250 g of trichlorphon 80% w/v, 200 mL ofchlorfenvinphos 24% w/v and 200 mL of endosulfan35% w/v. All products were applied by knapsacksprayers onto the tree, directly to the foliage. Theamount of liquid sprayed was about 1 L per tree.

Olive processingThe olives were harvested in mid-December andimmediately processed into oil employing a scalemodel of an oil mill (Abencor system, MC2 Ingenierıay Sistemas, SL, Seville, Spain). This scale model wascomposed of a hammer mill, a thermo-mixer and acentrifugal separator. After crushing, the paste wasmixed and the mix was decanted to separate the waterfrom the oil.

ReagentsPesticide standards were obtained from Chem Ser-vice Inc. (West Chester, PA, USA). As pre-treatment

prior to RPLC-GC analysis, the oil samples weremerely filtered through a 0.22 µm filter (Chromatog-raphy Research Supplies, Inc.). In the validationof endosulfan analytical method the spiked sampleswere obtained by dissolving each pesticide directly inpesticide-free olive oil: a weighed amount of pesticidewas dissolved in olive oil at a 1000 mg L−1 concen-tration. Afterwards, a stock solution 100 mg L−1 ofeach pesticide was prepared by mixing the initial con-centrated solutions containing the desired pesticidesand pesticide-free olive oil. Working pesticide solu-tions were prepared by diluting stock solution withpesticide-free olive oil. Methanol–water (70:30, v/v)was used as mobile phase. Methanol and water HPLCgrade were obtained from LabScan (Dublin, Ireland).Tenax TA 80–100 mesh (Chrompack, Middelburg,Netherlands), was used as packing material in theprogrammed temperature vaporizer (PTV) liner. Thepacked liner was conditioned under a helium streamby heating from 50 to 250 ◦C in 50 ◦C steps, holdingeach step for 10 min. The final temperature of 250 ◦Cwas maintained for 60 min.

InstrumentationRPLC-GC was performed by online coupled equip-ment consisting of an HPLC and a GC equippedwith the TOTAD interface. The HPLC system wascomposed of a manual injection valve (model 7125,Rheodyne, Rohnert Park, CA, USA) provided with a20 µL loop; a vacuum desgasifier (HP model 1100); aquaternary pump (HP model 1100); a column oven(HP model 1100) and a diode-array ultraviolet (UV)detector (Perkin-Elmer model LC 235). Two gas chro-matographs were employed. Both were Konik 4000B(Konik, Sant Cugat de Valles, Barcelona, Spain) andboth were equipped with a TOTAD interface (USPatent 6 402 947 B1, exclusive right assigned toKonik-Tech, Sant Cugat del Valles, Barcelona, Spain).One of them was equipped with an NPD and the otherone with an ECD. KoniKrom 32 (Konik, Sant Cugatde Valles, Barcelona, Spain) software was used toobtain data from LC and GC runs and to automatethe process. The TOTAD interface operation modehas been described elsewhere.9–11

The LC column used was a 50 × 4.6 mm i.d.column packed with modified silica (C4, Kromasil100-10, Hichrom, Berks, UK). The GC columnwas a Quadrex (Weybridge, UK) fused-silica column(30 m × 0.32 mm i.d.) coated with 5% phenyl methylsilicone (film thickness 0.25 µm).

Pesticide analysisOrganophosphorus pesticides were analysed byRPLC-GC-NPD using the TOTAD interface withthe method recently developed by our research group.8

Chromatographic conditions were as follows.

LC conditionsThe initial flow rate (2 mL min−1) was maintaineduntil pesticide elution began. When the beginning

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R Sanchez et al.

of the fraction of interest reached the six-port valve,this was automatically switched and the pump flowwas changed from 2 to 0.1 mL min−1. This flow wasmaintained during the LC-GC transfer step. After thetransfer, the flow was changed back to 2 mL min−1

and the gradient was raised to 100% methanol within1 min and maintained for at least 20 min in order toelute all the olive oil compounds from the LC column.

LC-GC transfer conditionsInitially, the interface and GC oven temperatureswere stabilized at 100 ◦C and 40 ◦C, respectively. Thecarrier gas (helium) flow entered the packed linerthrough the oven side and through the opposite side,both at 500 mL min−1.

The solvent eluted before the fraction of interest wassent to waste. When the front of the pesticide fractionreached the six-port valve, this was automaticallyswitched, transferring the fraction to the GC, withan LC flow rate of 0.1 mL min−1. The volume ofthe LC fraction containing the organophosphoruspesticides was 2 mL, so that the transfer step took20 min. During LC-GC transfer, the analytes wereretained on the packed material in the liner, while thesolvent was vented to waste. When the transfer stepwas completed, the six-port valve was automaticallyswitched, so that the LC eluent was sent to waste.

An additional purge time (3 min) was established tototally eliminate the remaining solvent from the glassliner and from the transfers tubing. Temperature andhelium flow were maintained constant for this time.

GC conditionsAfterwards, the valves and pneumatic controllerswere changed in such a way that helium enteredonly through the usual gas inlet to a PTV injectorat 1.8 mL min−1 and exited only through the GCcolumn. The TOTAD interface was heated to 300 ◦Cand maintained for 5 min at this temperature in orderto achieve thermal desorption of the retained analytes,which were transferred to the capillary column.

The column temperature was maintained at 40 ◦Cfor 1 min, raised to 170 ◦C at 20 ◦C min−1, thento 210 ◦C at 3 ◦C min−1, and finally to 230 ◦C at5 ◦C min−1. The final temperature was maintained for15 min. The NPD temperature was kept at 275 ◦C.

Endosulfan was analysed by RPLC-GC-ECD,using the method developed in the present work.Chromatographic conditions were the same as thoseindicated earlier for the analysis of organophosphoruspesticides, except the selected LC fraction of interest.ECD temperature was kept at 300 ◦C. Validation ofthe analytical method is presented in the Results andDiscussion section.

Statistical analysisStatistical analysis was performed with SPSS 11.0software.

RESULTS AND DISCUSSIONThe first step in the endosulfan analytical methodwas to fix the LC fraction in which this pesticidewas eluted. For this purpose, an olive oil samplespiked with endosulfan at a high concentration(50 mg L−1) was sampled. LC conditions were thesame as for the organophosphorus analytical method.8

The endosulfan eluted between 0.90 and 1.85 min,resulting in a volume of 1.9 mL, which was thefraction transferred from the LC to GC. Then,20 µL of olive oil spiked with 1 mg L−1 of both α

and β isomers and endosulfan sulfate were injectedand analysed by RPLC-GC-ECD. Both endosulfanisomers and endosulfan sulfate, previously identifiedby sampling a standard solution of each one, werecompletely separated by GC, as can be observed inFig. 1(a). The specificity of the proposed methodwas assessed by analysing a control blank oliveoil sample (Fig. 1b). The absence of peaks at theretention time of the endosulfan peaks showed that nointerference occurred. The limits of detection (LODs)were calculated as the amount giving a signal equal tofive times the background noise, whereas the limits ofquantification (LOQs) were determined considering avalue of 10 times the background noise. The LODsand LOQs are indicated in Table 1. The linearity ofthe method was determined using olive oil samplesfortified in the range 0.1–1 mg L−1 and consideringthe absolute peak areas. It must be stressed that nointernal standard was necessary. The detector responsewas linear in the range of concentrations studied.Determination coefficients are indicated in Table 1.The repeatability of the method was determined byrepeating five times the analysis of an olive oil samplefortified at two different concentrations (0.1 and1 mg L−1). The relative standard deviations (RSD) forthe absolute peak areas were lower than 9% (Table 1).The reproducibility of the analytical procedure wasalso determined by replicate analysis of the fortifiedsamples on five different days. The RSD are indicatedin Table 1.

The developed analytical method was applied to thedetermination of endosulfan in real olive oil samplesobtained from olives treated with the pesticide, asindicated in the Materials and Methods section.Although the residue level of each isomer and ofendosulfan sulfate could be quantified separately, thesum is considered in the study. Organophosphoruspesticides were analysed by RPLC-GC-NPD.8

Figure 2 shows the gas chromatogram correspond-ing to the RPLC-GC-NPD analysis of organophos-phorus pesticides in an olive oil sample obtained fromolive trees treated with the mixture of the pesticides inJuly and November at the recommended doses. Nei-ther trichlorphon nor chlorfenvinphos residues weredetected in any of the olive oil samples analysed.Trichlorphon was not detected, probably because ofits high water solubility (120 g L−1).12 When a solu-tion of trichlorphon in methanol was injected into theLC, the pesticide peak could be identified in the GC

1928 J Sci Food Agric 86:1926–1931 (2006)DOI: 10.1002/jsfa

Analysis of pesticide residues in olive oil. Part II

Table 1. Limits of quantification (LOQ); limits of detection (LOD), determination coefficients (R2) (concentration range from 0.1 to 1 mg L−1) and

relative standard deviation (RSD) (n = 5) at two different concentrations and on five different days

RSD (%)

LOQ (µg L−1) LOD (µg L−1) R2 (1 mg kg−1) (0.1 mg kg−1) Different days

α-Endosulfan 15 7 0.998 9.07 2.72 6.92β-Endosulfan 15 7 0.995 2.38 4.24 10.07Endosulfan sulfate 134 67 0.997 7.72 8.82 14.05

0 5 10 15 2520

(a)

(b)

Time (min)

α-en

dosu

lfan

β-en

dosu

lfan

endo

sulfa

n su

lfate

Figure 1. GC chromatograms obtained in the RPLC-GC-ECDanalysis of (a) an olive oil sample fortified at 1 mg L−1 and (b) blanktrace. Endosulfan peaks are indicated in the figure. Conditions areindicated in the Materials and Methods section.

chromatogram. Nevertheless, when a sample of oliveoil spiked with trichlorphon was analysed, the pesti-cide peak did not appear in the gas chromatogram,which suggests that trichlorphon is not soluble in theolive oil. It can be concluded therefore that this water-soluble pesticide passes into the aqueous phase during

the extraction of oil from olives, as indicated by otherauthors.3 Similar results were obtained in a previousstudy with dimethoate, which is also a water-solublepesticide.6

Chlorfenvinphos could not be identified in any ofthe olive oil samples, but was quantified in an oliveoil sample spiked with 1 mg L−1 of this pesticide.This pesticide is presumably rapidly degraded andno residues could be found higher than the LOD(28 µg L−1) in any of the obtained samples.

The other three pesticides used (malathion, diazi-non and endosulfan) are fat-soluble and so theirresidues tend to concentrate in the oil. None ofthe samples of oil made from olives treated only inJuly showed malathion residues but all of them haddiazinon and endosulfan residues higher than theirrespective LODs. In a previous work6 no pesticideresidues were found higher than the LODs in anysamples obtained from olives treated only in summer.It must be pointed out that the use of selective detec-tors instead of an FID in this study allowed muchlower LODs to be achieved.

All the oil samples made from olives treated in Julyand November, both at the recommended dose andat twice the dose, showed diazinon, endosulfan andmalathion residues higher than their respective LODs.

The pesticide residues in the oil made from olivesthat were treated at the recommended doses oncein the year were compared with those that receivedtwo treatments. As the samples are independent,we first performed the Levene test to study whetherthe variances of the populations could be consideredequals. This was followed by Student’s t-test of equalmeans for independent samples. The Levene testresults indicated that the variances can be consideredequals and the results of Student’s t-test are shownin Table 2. It can be seen that this test provided aP-value lower than 0.05 in all cases, so the differencesbetween means were statistically significant and theolives with two treatments in July and November left

Table 2. Difference between average obtained from olives treated twice (July and November) and once only (July)(X2 − X1)

PesticideNumber of samples

(July)Number of samples(July–November)

(X2 − X1)

(mg L−1)

Standarderror P-value

Diazinon 8 8 1.99 0.49 0.00Malathion 8 8 0.27 0.52 0.00Endosulfan 8 8 0.29 0.85 0.023 pesticides 24 24 0.84 0.17 0.00

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0 10 20

Dia

zino

n

Mal

athi

on

Time (min)

Figure 2. GC chromatogram of an olive oil sample obtained from the direct LC-GC-NPD analysis of the same olive oil sample of Fig. 1. Residuefound in the oil 1.9 mg L−1 of diazinon and 0.4 mg L−1 of malathion. Scale: 200 mV.

more residues in the oil than those given one treatmentin July.

To study whether the differences observed in thethree pesticide concentrations detected in the sampleswere statistically significant (with a significance levelof 0.05 in all the studies), an analysis of variance wasperformed, the results of which are shown in Table 3.As the P-value is lower than 0.05, we rejected thenull hypothesis of equal averages and concluded thatthe differences between the residues were significant.A post hoc analysis was then made to compare eachpair of pesticides. The results are shown in Table 4.The LSD (least significant difference) test, basedon Student’s t-test, suggested that the differencesbetween diazinon and each of the other two pesticideswere statistically significant (P-value = 0.00 in bothcases), and the difference between endosulfan andmalathion was also significant (P-value = 0.03). Fromthis study we concluded that malathion providedthe lowest level of pesticide residues, followed byendosulfan and diazinon. The use of endosulfan hasbeen banned by Spanish law, and consequently cannotbe recommended. If we had to use one of the studiedpesticides, trichlorphon or clorphenvinphos might beconsidered the most appropriate, because neither leftresidues, followed by malathion and finally diazinon.

Although endosulfan cannot be legally used inSpain, this study was interesting in order to testwhether this forbidden pesticide had in fact beenused. The experiment carried out shows that thedeveloped analytical method detected endosulfan even

Table 3. Comparison of average pesticide residues (X) by ANOVA

PesticideNumber ofsamples X (mg L−1) F-ratio P-value

Diazinon 24 1.44Malathion 24 0.13 20.47 0.00Endosulfan 24 0.59

Table 4. Difference between average residue levels of each pair of

pesticides (Xi − Xj)

Pair of pesticides(Xi − Xj)

(mg L−1)

Standarderror P-value

Diazinon–malathion 1.31 0.21 0.00Diazinon–endosulfan 0.84 0.21 0.00Endosulfan–malathion 0.47 0.21 0.03

when it had been applied only in summer. When thedeveloped method was used to analyze real olive oilfrom Castilla–La Mancha, endosulfan residues werefound in some of them. Lentza-Rizos analysed 338virgin olive oil samples from Greece and 22% of thesamples contained endosulfan residues although atvery low levels (from 0.02 to 0.57 mg kg−1).13

To ascertain whether the careless use of pesticidesin the field leads to pesticide residues in the olive oilhigher than the MRLs, we took into considerationthe results obtained from the olive oil made fromolives that were treated in July and November withthe normal and double doses. The normality ofthe populations was confirmed. The Levene testresults indicated that the variances can be consideredequal except for the case of diazinon. The resultsof the Student’s t-test of equal means, assumingequal variances except for the case of diazinon, areshown in Table 5. As expected, the double-dosetreatments left more residues that the normal doses.The differences between means were statisticallysignificant for endosulfan, malathion, diazinon andthe total of the three pesticides (P-value lower than0.05). In any case, malathion residues were lower than0.5 mg L−1 even when a double dose had been used totreat the trees. The diazinon residues found in the oilobtained from olives treated at the recommended dosewere always lower than the MRL (2 mg L−1 for virginoil)2 and it was slightly higher in the oil obtainedfrom olives treated at twice the recommended dose

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Analysis of pesticide residues in olive oil. Part II

Table 5. Difference between average pesticide residues in oil obtained from olives treated with twice the recommended and the recommended

application dose(XT − XR)

PesticideNo. of samples

(twice)No. of samples(recommended)

(XT − XR)

(mg L−1)

Standarderror P-value

Diazinon 8 8 0.29 0.10 0.01Malathion 8 8 0.16 0.06 0.01Endosulfan 8 8 1.17 0.16 0.003 pesticides 24 24 0.54 0.25 0.02

(2.9 mg L−1). Bearing in mind that endosulfan cannotbe used in Spain, malathion is preferable to diazinonbecause, even when used at double the dose, theresidues found in the oil were lower.

CONCLUSIONA fully automated RPLC-GC-ECD method foranalysing endosulfan in olive oil has been developed.The method allows quantification of α-endosulfan,β-endosulfan and endosulfan sulfate separately andgives good linearity and repeatability. This method canbe used to detect practices forbidden in Spain evenwhen endosulfan is used only in summer treatment.

Neither trichlorfon nor chlorfenvinphos residueswere found in the oil. Trichlorfon was not foundowing to its high water solubility, and chlorfenvinphospresumably because it degrades quickly. The differ-ences between pesticide residues for the three otherpesticides used in the present study were significant.Malathion provided the lowest residues, followed byendosulfan and diazinon. Malathion residues werenever higher than 0.5 mg L−1 even when twice therecommended doses were used to treat the olivetrees.

ACKNOWLEDGEMENTSFinancial support by Comision Interministerial deCiencia y Tecnologıa (CICYT), InterministerialCommission of Science and Technology, Spain,project PTR 1995-0626-OP is gratefully acknowl-edged. Raquel Sanchez Santiago thanks the Consejerıade Ciencia y Tecnologıa of the Junta de Comunidadesde Castilla–La Mancha and the European Social Fundfor her grant. The authors thank Pedro Hormigos fromValseco SL (Cebolla, Toledo, Spain), who carried outthe experimental treatments in the olive grove.

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